Insulin signals through several intracellular pathways. Here, we tested the hypothesis that insulin modulates Na⁺/H⁺-exchange activity in vascular cells through H₂O₂-mediated inhibition of tyrosine phosphatase SHP-2. We measured intracellular pH (pH_i) in isolated mouse mesenteric arteries using fluorescence confocal and wide-field microscopy. In the absence of CO₂/HCO₃^-, removal of bath Na⁺ produced endothelial acidification (pH_i=-0.71±0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state pH_i (pH_i=-0.28±0.08). Insulin and H₂O₂ acidified endothelial cells 0.2-0.3 pH-units and reduced the acidification upon Na⁺-removal by ~65%. Cariporide abolished the effect of insulin and H₂O₂. In vascular smooth muscle cells, H₂O₂ produced intracellular acidification (pH_i=-0.48±0.06) as did high concentrations of insulin (pH_i=-0.03±0.01). Na⁺/H⁺-exchange activity after an NH₄⁺-prepulse was ~80% attenuated by H₂O₂ and ~40% by high insulin concentrations. H₂O₂ had no effect on Na⁺,HCO₃^--cotransport activity. NHE1 (slc9a1) was the only plasma membrane NHE isoform detected in mouse mesenteric arteries by RT-PCR analyses. In both cell types, polyethylene glycol catalase abolished the effect of insulin on pH_i. Exposure to insulin increased the intracellular concentration of reactive oxygen species estimated with the fluorophore 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein. The SHP-2 selective inhibitors NSC-87877 and PTP inhibitor IV reduced steady-state pH_i up to 0.3 pH-units and inhibited Na⁺/H⁺-exchange activity 60-80%; when applied in combination with insulin or H₂O₂, no further effect was obtained. We conclude that Na⁺/H⁺-exchange contributes to pH_i regulation in arterial endothelial and smooth muscle cells in situ and is inhibited by insulin and H₂O₂. We propose that insulin signaling involves H₂O₂ and inhibition of protein tyrosine phosphatase SHP-2.